Evidence for the Plausibility
of anEndosymbiotic Origin of Eukaryotic
Organelles

In her book The Origin of Eukaryotic Cells, microbiologist Lynn
Margulis outlined her hypothesis that eukaryotic cells (cells with
nuclei, mitochondria, and sometimes chloroplasts) originated from the symbioses
of prokaryotic cells. This is now known as the endosymbiotic theory
for the origin of eukaryotic cells. Student Joshua
A. Bond has done a very nice introduction to Margulis' Endosymbiotic
Theory. Another, slightly more technical discussion can be found at
Endosymbiosis
and The Origin of Eukaryotes from Kimball's
Biology Pages (an excellent resource for biological information).

The purpose of this page is to present some intriguing evidence supporting
Margulis' endosymbiotic theory that is mentioned by Josh in his paper.
Microbiologist Kwang Jeon has spent the last two or three decades working
with strains of Amoeba proteus that have been infected with bacteria. In
some cases, although many of the infected amoeba were killed by the infection,
a few survived with populations of the bacteria remaining alive within
the amoebas' cells. Over time, some of these surviving but infected amoebae
became dependent on the bacteria within their cells ( K.W.
Jeon 1972 , K.W. Jeon 1987 ).

Kwang and others showed that the amoebae could not survive without the
internal bacteria using surgical methods ( K.W.
Jeon 1972 , K.W. Jeon et al.
1976 ) . These methods consisted of removing the nucleus of an infected
cell and placing it into another cell that had previously had its nucleus
removed. A nucleus from an infected amoeba would only survive if the cell
it was inserted into had the bacterial endosymbionts in its cytoplasm.
If the endosymbionts were not present, the previously infected nucleus
(and hence the entire cell) died.

Kwang also showed that the infected amoeba required the endosymbiotic
bacteria using chemical methods ( K.W.
Jeon et al. 1977 ). This was done by treating the infected amoebae
with the antibiotic chloramphenicol
(CAP). The antibiotic reduced the number of bacteria living in the amoeba
to less than 10% of the level found in control lines (not treated with
CAP). None of the amoebae that had all of their endosymbiotic bacteria
killed were able to survive. (Note that the amoeba's mitochondria
were damaged by the CAP, but at least some of the amoebae retaining their
symbionts were able to survive, whereas none of the amoebae that lost all
of their endosymbiotic bacteria survived.)

Further evidence for the amoebae's dependence on the endosymbiotic bacteria
was provided by K.W. Jeon 1995 and J.Y.
Choi et al. 1997 . The researchers demonstrated that infected amoebae
no longer produced a protein (an enzyme in this case) required for survival.
However, the infected amoebae had some activity for that enzyme (about
half the level found in normal, uninfected amoebae). It was shown that
it was the bacteria providing the enzyme that the amoeba was no longer
producing. Furthermore, if the bacteria were removed from the amoebae,
the amoebae's nucleoli were damaged, apparently due to the loss of the
enzyme formerly provided by the bacteria. It is interesting that the amoebae
were no longer able to produce the enzyme because of interference from
the infecting bacteria, but because the amoebae would be damaged if the
bacteria were removed, the endosymbiotic bacteria were now required for
the amoebae's survival.

Although none of this provides proof that the origin of the eukaryotic
cell was due to endosymbiosis, it has been demonstrated that such an event
can happen naturally even with modern cells. This is evidence for
the plausibility of Margulis' endosymbiotic theory of the origin of eukaryotic
cells.

A strain of large, free-living amoeba that became dependent on bacterial
endosymbionts which had infected the amoebae initially as intracellular
parasites, was studied by micrurgy and electron microscopy. The results
show that the infected host cells require the presence of live endosymbionts
for their survival.Thus, the nucleus of an infected amoeba can form a viable
cell with the cytoplasm of a noninfected amoeba only when live endosymbionts
are present. The endosymbiotic bacteria are not digested by the host amoebae
and are not themselves used as nutritional supplement. While the host amoebae
are dependent specifically on the endosymbionts, the latter can live inside
amoebae of different strains, indicating that their dependence on the host
cells is not yet strain specific.

J Protozool 1977 May;24(2):289-93
Effect of chloramphenicol on bacterial endosymbiotes in a strain of Amoeba
proteus.

Jeon KW, Hah JC

The effect of chloramphenicol (CAP) on the bacterial endosymbiotes of
a strain of Amoeba proteus was studied by growing the symbiotic amebae
in media containing 0.5-1.6 mg/ml CAP for up to 4 weeks. Treatments with
CAP caused such ultrastructural changes as expansion of the nuclear zone
and deformation of symbiotes. The CAP treatment also damaged the mitochondria,
e.g. disappearance of central and protrusion of peripheral cristae. Number
of bacteria per ameba decreased to less than 10% of control in CAP-containing
media, but no viable amebae became completely free of symbiotes. The results
supported previous studies that amebae were dependent on endosymbiotes.

A strain of nonsymbiotic A. proteus was infected with endosymbiotic
bacteria isolated from another strain of amoeba which had become dependent
on the symbionts after a few years of spontaneously established symbiosis.
In the newly infected amoebae, the bacteria avoided digestion and multiplied
at a faster rate than the hosts, reaching the maximum carrying number (about
42,000 per amoeba) in fewer than ten cell generations of the hosts. The
experimentally infected amoebae were also examined under the electron microscope,
and the development of bacteria-containing vesicles was followed. The results
show that the infective bacteria that were initially harmful to host amoebae
have become harmless and that they have changed in their mode of multiplication
during the course of establishing a stable symbiosis with their hosts.

The large, free-living amoebae have been widely used as model cells for
studying a variety of biological phenomena, including cell
motility, nucleocytoplasmic interactions, membrane function, and symbiosis.
Results of studies by our group on amoebae as moving
cells, as material for micrurgical manipulations, and as hosts for intracellular
symbionts are summarized here. In particular, our recent
studies of the amoeba as a microcosm, in which spontaneously infecting
foreign microbes have become integrated as necessary cell
components, are described in some detail. These processes have involved
an initial microbial infection, mutual adaptation by the host
and symbionts, and development of obligatory symbiosis. Evidence is presented
to show that symbiont-derived macromolecules are
involved in the protection of symbionts from digestion, the symbionts have
acquired regulatory elements on their chromosomal genes to
enhance production of beneficial gene products, and symbionts apparently
utilize host-derived macromolecules to their benefit. These
studies involved morphological observations both at light and electron
microscopic levels, physiological and genetic studies, production
and use of poly- and monoclonal antibodies, and molecular-biological approaches
including gene cloning and sequencing. It is shown
that amoebae are uniquely suited as model cells with which to study these
phenomena.

Symbiont-bearing xD amoebae no longer produce a 45-kDa cytoplasmic protein
that functions as S-adenosylmethionine synthetase in symbiont-free D amoebae.
The absence of the protein in xD amoebae is attributable to xD amoeba's
failure to transcribe the corresponding gene as a result of harboring bacterial
symbionts. However, xD amoebae have about half the level of enzyme activity
found in D amoebae, indicating that they use an alternative source for
the enzyme. xD amoebae originated from D amoebae by bacterial infection
and now depend on their symbionts for survival. xD amoebae exhibit irreversible
nucleolar abnormalities when their symbionts are removed, suggesting that
X-bacteria supply the needed enzyme. A monoclonal antibody against the
45-kDa protein was produced and used as a probe in cloning its corresponding
cDNA. The product of the cDNA was found to have S-adenosylmethionine synthetase
activity. These results show how symbiotic X-bacteria may become essential
cellular components of amoeba by supplementing a genetic defect for an
amoeba's house-keeping gene that is brought about by an action of X-bacteria
themselves. This is the first reported example in which symbionts alter
the host's gene expression to block the production of an essential protein.

J Eukaryot Microbiol 1997 Nov-Dec;44(6):614-9

A symbiont-produced protein and bacterial symbiosis in Amoeba proteus.

Pak JW, Jeon KW.

Department of Biochemistry, University of Tennessee, Knoxville, USA.

Gram symbiotic X-bacteria present in the xD strain of Amoeba proteus as
required cell components, synthesize and export a large
amount of a 29-kDa protein (S29x) into the host's cytoplasm across bacterial
and symbiosome membranes. The S29x protein
produced by E. coli transformed with the s29x gene is also rapidly secreted
into the culture medium. Inside amoebae, S29x enters the
host's nucleus as detected by confocal and immunoelectron microscopy, although
it is not clear if S29x is selectively accumulated inside
the nucleus. The deduced amino-acid sequence of S29x has a stretch of basic
amino acids that could act as a nuclear localization
signal, but there is no signal peptide at the N-terminus and the transport
of S29x is energy independent. The functions of S29x are not
known, but in view of its prominent presence inside the amoeba's nucleus,
S29x is suspected to be involved in affecting the expression
of amoeba's nuclear gene(s).